Project Summary Carbon-halogen bonds are ubiquitous in agrochemicals1 and pharmaceutical compounds at every stage in development.2 Although much attention is paid to the unique properties of the carbon-fluorine bond,3-6 carbon-chlorine bonds are in fact more numerous within FDA-approved drugs.2 The most numerous type of C?Cl bond in pharmaceuticals by far is the aryl C?Cl bond.7 Traditional chemistry for forming aryl C?Cl bonds, such as electrophilic halogenation and halogen exchange with organometallic arenes, are harsh and suffer poor functional group tolerance.8 Modern transition metal-catalyzed C?H halogenations8 can still be challenging, and the electronic preferences of the substrate can only be overcome with elaborate directing groups.9 Enzymatic catalysis, in contrast, are able to exert remarkable regiocontrol in aryl C?Cl bond formation. Flavin-dependent halogenases (FDHs)10,11 are able to halogenate a variety of electron-rich (hetero)arenes, such as phenols,12 pyrroles,13 and indoles14 via an electrophilic aromatic substitution mechanism by a lysine chloramine intermediate.15 The tryptophan halogenases, which halogenate the indole of the tryptophan sidechain, perhaps offer the most detailed glimpse into how highly tunable this class of catalyst is. Different tryptophan FDHs have been discovered that are able to halogenate at either the 5-16, 6-17, or 7-position14,18 of tryptophan, overriding the electronic preferences of the indole for C2-halogenation. The diverse regioselectivity in this set of homologous enzymes hints at evolvability toward new function not found in nature.19 One FDH, RebH, has been subject to extensive engineering efforts, including directed evolution for thermostability,20 altered regioselectivity,21 and expanded substrate scope.22,23 Despite these accomplishments, the engineered RebH variants remain unable to halogenate electron-deficient arenes, and alternative halogenase biocatalyst discovery strategies that may further expand substrate scope remain largely unexplored. The aim of the proposed research is to extend FDH halogenation to electron-deficient arenes, characterize new halogenases from genetic sequence databases, and extend halogenase activitity to large, biologically relevant molecules. Specifically: 1a) I will use directed evolution to establish and extend the limit of arene electron deficiency accessible to RebH-catalyzed halogenation; 1b) I will use rational active-site mutagenesis and amber codon suppression to incorporate electron-deficient lysine residues within the RebH active site to extend halogenation activity to particularly electron-deficient heteroarenes such as unactivated quinolines; 2) I will mine genetic databases for putative FDHs, characterize their halogenase activity on a library of structurally diverse arenes, and link the discovered substrate scope to phylogeny using bioinformatics and to structure using X-ray crystallographic analysis; and 3) In collaboration with Prof. Scott Snyder, I will use directed evolution of the newly discovered FDHs to halogenate large, structurally complex resveratrol oligomers. The sum of the research proposed will improve our understanding of FDH catalysis as well as generate useful catalysts for functionalizing molecules important in medicinal chemistry.